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Syndapin is dispensable for synaptic vesicle endocytosis at the Drosophila larval neuromuscular junction.

Kumar V, Alla SR, Krishnan KS, Ramaswami M - Mol. Cell. Neurosci. (2008)

Bottom Line: The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult "escapers" frequently emerge.Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis.Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Smurfit Institute of Genetics and Trinity College Institute of Neuroscience, Lloyd Building, University of Dublin, Trinity College, Dublin 2, Ireland. kumarv@tcd.ie

ABSTRACT
Syndapin is a conserved dynamin-binding protein, with predicted function in synaptic-vesicle endocytosis. Here, we combine genetic mutational analysis with in vivo cell biological assays to ask whether Drosophila syndapin (Synd) is an essential component of synaptic-vesicle recycling. The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult "escapers" frequently emerge. Contrary to expectation, we report that the Synd protein is predominantly postsynaptic, undetectable at presynaptic varicosities at Drosophila third-instar larval neuromuscular junctions. Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis. Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

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Conserved structure and biochemical interactions of Drosophila syndapin. (A) Like its mammalian orthologs, Drosophila syndapin has an N-terminal F-BAR domain comprising FCH and coiled-coil (CC) regions and a C-terminal SH3 domain. (B) Synd interacts with proline-rich domain of shibire. Bacterially expressed GST-shibire PRD pulls down syndapin from Drosophila head lysates, as seen in western blot analysis of pulled down proteins probed with anti-Synd antibody. (C) Various domains of syndapin were expressed as GST-tagged fusion protein and binding proteins in fly head lysate pulled down using Glutathione sepharose beads. Western blot of pulled down proteins in each case were probed with anti-Wsp antibody. (D) Embryos (D1 and D2) and third instar larval ventral nerve cord (D3) of Drosophila stained with anti-Synd antibody. Note that Synd although enriched in central nervous system and ventral nerve cord, it is expressed ubiquitously in embryos. (E) Western blots of fly lysate from different stages of Drosophila development. A single band of about 57 kDa was observed at all stages analyzed.
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fig1: Conserved structure and biochemical interactions of Drosophila syndapin. (A) Like its mammalian orthologs, Drosophila syndapin has an N-terminal F-BAR domain comprising FCH and coiled-coil (CC) regions and a C-terminal SH3 domain. (B) Synd interacts with proline-rich domain of shibire. Bacterially expressed GST-shibire PRD pulls down syndapin from Drosophila head lysates, as seen in western blot analysis of pulled down proteins probed with anti-Synd antibody. (C) Various domains of syndapin were expressed as GST-tagged fusion protein and binding proteins in fly head lysate pulled down using Glutathione sepharose beads. Western blot of pulled down proteins in each case were probed with anti-Wsp antibody. (D) Embryos (D1 and D2) and third instar larval ventral nerve cord (D3) of Drosophila stained with anti-Synd antibody. Note that Synd although enriched in central nervous system and ventral nerve cord, it is expressed ubiquitously in embryos. (E) Western blots of fly lysate from different stages of Drosophila development. A single band of about 57 kDa was observed at all stages analyzed.

Mentions: Drosophila syndapin (synd) was identified by analysis of the Drosophila genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis or endocytosis of synaptic vesicles (Lloyd et al., 2000). Unlike mammals, which have three genes for syndapin, the Drosophila genome contains a single gene for syndapin (Supplementary Fig. S1A). The synd locus produces a single transcript of about 3.2 kb comprising 10 exons. The Drosophila syndapin shares an overall sequence similarity of about 55% to its mammalian orthologs (Supplementary Fig. S1B). Conceptual translation of this experimentally confirmed synd ORF predicts a protein of 494 amino acids with domain organization remarkably similar to vertebrate syndapins (Fig. 1A): thus, it consists of the N-terminal F-BAR domain with about 49% identity and a C-terminal SH3 domain with over 65% identity to mammalian syndapin 1.


Syndapin is dispensable for synaptic vesicle endocytosis at the Drosophila larval neuromuscular junction.

Kumar V, Alla SR, Krishnan KS, Ramaswami M - Mol. Cell. Neurosci. (2008)

Conserved structure and biochemical interactions of Drosophila syndapin. (A) Like its mammalian orthologs, Drosophila syndapin has an N-terminal F-BAR domain comprising FCH and coiled-coil (CC) regions and a C-terminal SH3 domain. (B) Synd interacts with proline-rich domain of shibire. Bacterially expressed GST-shibire PRD pulls down syndapin from Drosophila head lysates, as seen in western blot analysis of pulled down proteins probed with anti-Synd antibody. (C) Various domains of syndapin were expressed as GST-tagged fusion protein and binding proteins in fly head lysate pulled down using Glutathione sepharose beads. Western blot of pulled down proteins in each case were probed with anti-Wsp antibody. (D) Embryos (D1 and D2) and third instar larval ventral nerve cord (D3) of Drosophila stained with anti-Synd antibody. Note that Synd although enriched in central nervous system and ventral nerve cord, it is expressed ubiquitously in embryos. (E) Western blots of fly lysate from different stages of Drosophila development. A single band of about 57 kDa was observed at all stages analyzed.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2697329&req=5

fig1: Conserved structure and biochemical interactions of Drosophila syndapin. (A) Like its mammalian orthologs, Drosophila syndapin has an N-terminal F-BAR domain comprising FCH and coiled-coil (CC) regions and a C-terminal SH3 domain. (B) Synd interacts with proline-rich domain of shibire. Bacterially expressed GST-shibire PRD pulls down syndapin from Drosophila head lysates, as seen in western blot analysis of pulled down proteins probed with anti-Synd antibody. (C) Various domains of syndapin were expressed as GST-tagged fusion protein and binding proteins in fly head lysate pulled down using Glutathione sepharose beads. Western blot of pulled down proteins in each case were probed with anti-Wsp antibody. (D) Embryos (D1 and D2) and third instar larval ventral nerve cord (D3) of Drosophila stained with anti-Synd antibody. Note that Synd although enriched in central nervous system and ventral nerve cord, it is expressed ubiquitously in embryos. (E) Western blots of fly lysate from different stages of Drosophila development. A single band of about 57 kDa was observed at all stages analyzed.
Mentions: Drosophila syndapin (synd) was identified by analysis of the Drosophila genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis or endocytosis of synaptic vesicles (Lloyd et al., 2000). Unlike mammals, which have three genes for syndapin, the Drosophila genome contains a single gene for syndapin (Supplementary Fig. S1A). The synd locus produces a single transcript of about 3.2 kb comprising 10 exons. The Drosophila syndapin shares an overall sequence similarity of about 55% to its mammalian orthologs (Supplementary Fig. S1B). Conceptual translation of this experimentally confirmed synd ORF predicts a protein of 494 amino acids with domain organization remarkably similar to vertebrate syndapins (Fig. 1A): thus, it consists of the N-terminal F-BAR domain with about 49% identity and a C-terminal SH3 domain with over 65% identity to mammalian syndapin 1.

Bottom Line: The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult "escapers" frequently emerge.Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis.Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Smurfit Institute of Genetics and Trinity College Institute of Neuroscience, Lloyd Building, University of Dublin, Trinity College, Dublin 2, Ireland. kumarv@tcd.ie

ABSTRACT
Syndapin is a conserved dynamin-binding protein, with predicted function in synaptic-vesicle endocytosis. Here, we combine genetic mutational analysis with in vivo cell biological assays to ask whether Drosophila syndapin (Synd) is an essential component of synaptic-vesicle recycling. The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult "escapers" frequently emerge. Contrary to expectation, we report that the Synd protein is predominantly postsynaptic, undetectable at presynaptic varicosities at Drosophila third-instar larval neuromuscular junctions. Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis. Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

Show MeSH
Related in: MedlinePlus